Molecular epidemiology of Crimean-Congo hemorrhagic fever virus in Russia

In this report, we present new data on the diversity and geographical distribution of genetic variants in Crimean-Congo hemorrhagic fever virus (CCHFV) in Russia. Partial S, M, and L segment sequences of the CCHFV genome were obtained from 500 serum samples from CCHF patients and 103 pools of ticks collected in the south of the European region of Russia in 2007–2017. The investigated viral strains belonged to the lineages Europe 1 (596 samples), Africa 3 (1 sample) and a new genetic lineage, Europe 3 (6 samples). The Russian CCHFV strains of the Europe 1 lineage formed four subgroups (Va-Vd) correlated with the geographical site of virus isolation. Segment reassortment events between strains of different subgroups within lineage Europe 1 were revealed. The complete S, M and L genome segments of 18 CCHFV strains belonging to different subgroups of the Europe 1 lineage and the complete S segments of 3 strains of the Europe 3 lineage and 1 strain of the Africa 3 lineage were sequenced. The analysis of the geographical distribution of CCHFV genetic variants in southern Russia revealed local viral populations with partially overlapping boundaries.

Introduction
Crimean-Congo hemorrhagic fever virus (CCHFV) is the etiological agent of Crimean-Congo hemorrhagic fever (CCHF), a severe disease in humans that can be transmitted via Ixodidae tick bites, contact with the tissues of infected animals, contact with the biological fluids of infected patients and vertical transmission [1]. It is endemic in the countries of Africa, Asia, and southeast Europe and in a number of regions in the south of the European region of Russia [2]. CCHFV belongs to the Orthonairovirus genus within the Nairoviridae family [3].
The epidemic situation of CCHF in Russia remains troublesome. In 1999-2020, 2,361 CCHF cases were detected in southern Russia, 94 of which (3.98%) had lethal outcomes. CCHFV is one of the most genetically diverse arboviruses [4]. The virus genome consists of three segments of single-stranded RNA with negative polarity: the S segment (1,670 bp), M segment (5,360 bp), and L segment (12,160 bp). The S segment encodes the nucleoprotein (482 aa); the M segment encodes the GPC polyprotein (1,688 aa)-a protein precursor of the Gn and Gc surface glycoproteins; and the L segment encodes the RNA-dependent RNA polymerase (3,945 aa) [5,6].
Based on an analysis of complete and partial S segment sequences, seven genetic lineages of CCHFV were distinguished and shown to be correlated with the geographical site of virus isolation; these lineages were Africa 1, Africa 2, Africa 3, Asia 1, Asia 2, Europe 1, and Europe 2 [7,8]. A phylogenetic analysis of partial and complete M and L segment sequences revealed nine and six genetic lineages, respectively, that were partially congruent with the S segmentbased lineages [1,[8][9][10]. Segment reassortment and recombination, which occur in simultaneous infections of vectors with viral strains belonging to different lineages and lead to the formation of new genetic variants, are characteristic of CCHFV [11][12][13].
Previous studies have established that CCHFV strains of the Europe 1 lineage are circulating in southern Russia in Stavropol Territory and the Rostov, Volgograd, and Astrakhan regions [14][15][16]. Strains from Turkey, southeast Europe, and Iran also belong to this genetic lineage [4,17]. Genetic differences between CCHFV strains of lineage Europe 1 circulating in various geographical regions have been revealed. The phylogenetic analysis of partial S segment sequences allows several subgroups within lineage Europe 1 to be separated. Russian CCHFV strains belong to three subgroups: Stavropol-Rostov-Astrakhan (Va), Volgograd-Rostov-Stavropol (Vb), Astrakhan-2 (Vc), and Crimea (Vd) [16,18,19]. Strains from the Balkans and Turkey form distinct subgroups [20][21][22].
This study aimed to obtain new data on the genetic diversity of CCHFV in Russia, to reveal prevailing genetic variants and to compare their spatial distribution.

Serum and tick samples
The materials used in this study were serum samples collected from CCHF patients in Russia in 2007-2017 that were positive for the presence of CCHFV RNA and pools of ticks belonging to the species Hyalomma marginatum, H. scupense, Rhipicephalus turanicus, Haemaphysalis punctata, and Dermacentor marginatus collected in southern Russia (Table 1).
Clinical samples of CCHF patients for whom site of infection was established were included in the study. The number of samples was correlated with the prevalence of CCHFV in each region. This study was approwed by the Stavropol Research Anti-Plague Institute's ethics committee. All CCHF patients personal data were analyzed anonymously.

Detection of viral RNA
Viral RNA was extracted from the clinical samples and tick suspensions using the RIBO-prep extraction kit (AmpliSens 1 , Russia), and complementary DNA was obtained using the REVERTA-L Kit (AmpliSens 1 , Russia). The detection of CCHFV in human sera and ticks was carried out using the RT-PCR CCHFV-FL Kit (AmpliSence 1 , Russia).

Genotyping
CCHFV genotyping was performed by sequencing three fragments of the viral genome: a partial S segment (position 115-652), partial M segment (position 4620-5075), and partial L segment (position 105-541), which were then subjected to phylogenetic analysis. The genetic variants present in different CCHFV strains were determined through the comparison of the corresponding cluster positions in phylogenetic trees based on partial S, M and L segments.
Fragments of the S, M and L segments were amplified using the primer pairs S 100f and S 680r [18]; 24 and 25 [23]; and L100f: gattggactcaggtgattgctggtc and L540r: gcctccctcgtgtctgtttc, respectively. The purification of the PCR products was carried out using the AxyPrep™ PCR Cleanup Kit (Axygen Biosciences, USA). Amplicons were bidirectionally sequenced using a Big Dye Terminator Kit v. 3.1 on an ABI 3130 Genetic Analyzer (Applied Biosystems, USA).

Complete genome sequencing
The complete S, M, and L segments of the CCHFV genome were amplified as 20 overlapping fragments using a set of primers (S1 Table). The concentration of the amplicons was assessed by performing electrophoretic separation in a 1% agarose gel. Equivalent amounts of each PCR product were combined, and the DNA was purified using the AxyPrep™ PCR Cleanup Kit (Axygen Biosciences, USA) and used to generate shotgun DNA libraries.

Phylogenetic analysis
Contigs were assembled using Newbler Assembler 2.9, Newbler Mapper 2.9 (454 Life Science), and Vector NTI 8.0 software. The nucleotide sequences of the partial and complete genome segments of CCHFV strains obtained in this study were compared with reference S, M and L segment sequences of CCHFV strains belonging to different genetic lineages available in Gen-Bank (S2 Table). The phylogenetic analysis of partial S, M and L segment sequences was conducted via the neighbor-joining method according to the Kimura-2 algorithm using Mega 5.05 software. Phylogenetic trees based on complete S, M and L genome segments were generated via the neighbor-joining method in Mega 5.05 software (Kimura-2 algorithm, statistical significance of the phylogenetic tree topology was assessed in the bootstrap test with 1000 replications) [24] and by using BEAST 2.0 (GTR+G model, strict clock, 10,000,000 generations) [25]. The MCC tree files were visualized using FigureTree 1.4.3

Geographic distribution analysis
The geographical distribution of genetic variants of CCHFV was analyzed using ArcGIS 10.1 software. The percentage of nucleotide differences between the strains of the Europe 3 lineage and the strains belonging to other lineages ranged from 13-15% for the S segment 24-25% for the M segment and 16,7-20,1% for the L segment. The phylogenetic trees based on the partial S and L segments confirmed the division of the Russian CCHFV strains into four subgroups within lineage Europe 1: a Stavropol-Rostov-Astrakhan (Va) strain, a Volgograd-Rostov-Stavropol (Vb) strain, an Astrakhan-2 (Vc) strain and a Crimea (Vd) strain (Fig 1A and 1C). The Balkan and Turkish strains were also separated into isolated groups. In the M segment tree, the Russian strains belonged to only three subgroups within the Europe 1 lineage: Va, Vb and Vd (Fig 1B).

Genetic diversity of CCHFV based on complete genome segments
The whole-genome sequencing of 18 CCHFV strains representing different genetic variants within the Europe 1 genetic lineage, along with the complete S segments of three strains of genetic lineage Europe 3 and one strain of genotype Africa 3, was performed. the full-length nucleotide sequences were submitted to GenBank and are included in the S1 File.
The obtained complete sequences of the S, M and L segments of Russian CCHFV strains were used for phylogenetic analysis. The phylogenetic analysis of the complete coding regions (ORFs) of the S, M and L segments was conducted by neighbor joining (Fig 2) and Bayesian methods (S2 File). The topology of the phylogenetic trees based on the partial S, M and L segment sequences and the complete segment sequences and constructed by different methods were similar. The phylogenetic analysis of the complete segment sequences

Territorial distribution of CCHFV genetic variants in Russia
The geographical distribution of the genetic variants of CCHFV in southern Russia was analyzed. The coordinates of the tick collection points and the infection sites of the CCHF patients established during the epidemiological analysis of cases were plotted on a map of the southern portion of the European region of Russia (S1 File).
The analysis of the geographical distribution of the genetic variants of CCHFV in southern Russia showed the presence of local viral populations with partially overlapping boundaries. CCHFV strains belonging to the Va-Va-Va genetic variant are circulating in the southern part of the CCHF natural focus and the Vb-Vb-Vb virus strains in the northern part (Fig 3A and 3B). Reassortant variants of CCHFV most often occur in the region of overlapping distribution areas of genetic variants Va-Va-Va and Vb-Vb-Vb (Fig 3C). Reassortant Vc-Vb-Va variants were revealed only in the Astrakhan region, and two isolates of the Vc-Va-Va genetic variant were found in the samples from Stavropol Territory. CCHFV strains belonging to subgroup Vd were detected only on Crimean Peninsula [19], and strains of the Europe 3 genetic lineage were detected only in the territory of the Republic of Kalmykia and Stavropol Territory. A single CCHFV strain of lineage Africa 3 was revealed east of Stavropol Territory (Fig 3D).
The prevalence of the circulating genetic variants of CCHFV in the administrative areas of the southern European part of Russia is presented in S1 Fig.

CCHF severity during infection by different CCHF virus genovariants in Russia
Clinical data (severity rate, the presence of hemorrhagic manifestations and clinical outcome) of patients infected with different genovariant of CCHF virus were analysed (Table 2).   The results showed risk severe clinical manifestation, hemorrhagic syndrome and fatal outcome increase for CCHFV by Europe-1 lineage (genetic variants Va-Vb-Va and Vb-Vb-Vb) compare with lineages Europe-3 and Africa-3. The small number of CCHF cases caused by CCHF virus, belonged to Europe-3 and Africa-3 lineages and some variants within genetic lineage Europe 1 is a limitation of the study.

Discussion
In the southern European part of Russia, the strains of lineage Europe 1 dominate the CCHFV population, and strains of this genotype are typical for this region. The Russian CCHFV strains of the Europe 1 genotype form four subgroups, correlated with the geographical sites of sample isolation. Previously it was shown that isolates from distinct regions of South Russia were intermixed [26], however analysis of geographical distribution of the genetic variants of CCHFV in southern Russia based on coordinates of virus isolates collection points confirmed the presence of local viral populations with partially overlapping boundaries. The evidence of segment reassortment between strains of different subgroups within lineage Europe 1 were revealed based on partial and complete genome sequences. Notably, all CCHFV strains belonging to subgroup Vc in the S segment phylogenetic tree, which were described in this work and are available in the GenBank database, were reassortant. Rarely, divergent isolates of Europe-1 were also found in Russia [26]. The separation of strains isolated from various regions of Russia, Europe and Turkey into different subgroups within the Europe 1 genetic lineage provides evidence of the independent evolution of CCHFV genetic variants circulating in local CCHFV populations formed in geographically remote and isolated areas. Due to the subsequent migration of infected ticks (the main vectors of CCHFV) on birds, the genetic variants of CCHFV gradually spread into new areas [27]. The circulation of several genetic variants in a region may lead to the formation of reassortant strains of CCHFV.
Isolates of the Africa 3 genotype and of the new genetic lineage Europe 3 were revealed for the first time in the studied region.
The identification of a CCHFV isolate belonging to the Africa 3 genotype in a serum sample from an CCHF patient from southern Russia (the patient did not leave the Stavropol territory and was bitten by ticks while gardening at home) confirms the possibility of the long-distance transport of CCHFV in infected ticks on migratory birds or livestock. However, no CCHFV strain of lineage Africa 3 has yet been detected in ticks collected from birds or livestock in Russia.
The detection of strains of the new genetic lineage Europe 3 provides new data on the genetic diversity of CCHFV in Russia and the world. The percentage of the detected strains belonging to lineage Europe 3 among the studied strains was extremely low (1%). Two cases of CCHF caused by virus, belonged to lineage Europe-3 were moderate severity, without hemorrhagical manifestation, with non-fatal outcome. Most likely, the Europe 3 lineage is a more ancient genetic lineage of CCHFV than the Europe 1 lineage. Strains of this lineage were first introduced into the territory of Russia and circulated in natural foci until they were replaced by strains of lineage Europe 1, which now predominates and causes 99.9% of CCHF cases recorded in Russia. Further studies of CCHFV strains belonging to this genetic lineage, including the evaluation of the geographical distribution of strains of this lineage in Russia and other countries and the sequencing of the complete M and L segments, are required. This will make it possible to estimate the genetic diversity and spatial distribution of the Europe 3 genetic lineage.


s2fieldsofstudy:
Biology
Medicine
Engineering
Materials Science

text:
Bioactive Cell-Derived ECM Scaffold Forms a Unique Cellular Microenvironment for Lung Tissue Engineering

Chronic lung diseases are one of the leading causes of death worldwide. Lung transplantation is currently the only causal therapeutic for lung diseases, which is restricted to end-stage disease and limited by low access to donor lungs. Lung tissue engineering (LTE) is a promising approach to regenerating a replacement for at least a part of the damaged lung tissue. Currently, lung regeneration is limited to a simplified local level (e.g., alveolar–capillary barrier) due to the sophisticated and complex structure and physiology of the lung. Here, we introduce an extracellular matrix (ECM)-integrated scaffold using a cellularization–decellularization–recellularization technique. This ECM-integrated scaffold was developed on our artificial co-polymeric BETA (biphasic elastic thin for air–liquid interface cell culture conditions) scaffold, which were initially populated with human lung fibroblasts (IMR90 cell line), as the main generator of ECM proteins. Due to the interconnected porous structure of the thin (<5 µm) BETA scaffold, the cells can grow on and infiltrate into the scaffold and deposit their own ECM. After a mild decellularization procedure, the ECM proteins remained on the scaffold, which now closely mimicked the cellular microenvironment of pulmonary cells more realistically than the plain artificial scaffolds. We assessed several decellularization methods and found that 20 mM NH4OH and 0.1% Triton X100 with subsequent DNase treatment completely removed the fibroblasts (from the first cellularization) and maintains collagen I and IV as the key ECM proteins on the scaffold. We also showed the repopulation of the primary fibroblast from human (without chronic lung disease (non-CLD) donors) and human bronchial epithelial (16HBE14o−) cells on the ECM-integrated BETA scaffold. With this technique, we developed a biomimetic scaffold that can mimic both the physico-mechanical properties and the native microenvironment of the lung ECM. The results indicate the potential of the presented bioactive scaffold for LTE application.

Introduction
Chronic respiratory diseases are one of the leading causes of death worldwide, for which symptom-relieving but no causal therapies exist [1]. This not only leads to a high burden of disease in the population, but also implies significant socio-economic costs [2]. Currently, lung transplantation is the only available causal therapy for chronic lung diseases, which is only available for end-stage disease [3]. The lungs are highly sophisticated to fully regenerate in the lab due to both their intricate architecture and complex function [4]. In the last decades, several strategies have been pursued to engineer a section of the lung tissue such as the alveolar-capillary barrier [5][6][7][8].
Artificial polymeric scaffolds based on synthetic and natural materials have been fabricated to imitate the extracellular matrix (ECM) in the lungs [7]. The scaffolds play a crucial role in tissue engineering (TE) development since they could be tailored to provide structural and physicomechanical support and emulate the cellular microenvironments to moderate cellular physiology [9,10]. Synthetic-based scaffolds such as poly(ε-)caprolactone (PCL) and polyethylene terephthalate (PET) have been manufactured to culture pulmonary epithelial cells [11][12][13][14][15]. On the other hand, natural-based scaffolds such as collagen (or its surrogate gelatin) and decellularized ECM (dECM) have shown excellent bioactivity and biocompatibility [7,16,17]. Nevertheless, the scaffolds based on synthetic and natural polymers suffer from a lack of cell affinity and poor physicomechanical properties, respectively [18,19]. Several studies have reported a composite of synthetic and natural polymers or hybrid scaffolds for lung tissue engineering (LTE) applications with a combination of biocompatibility and mechanical properties. [14,18,20]. However, the hybrid scaffolds failed to mimic the tissue-specific microenvironments.
We previously introduced the co-polymeric BETA (biphasic elastic thin for air-liquid culture conditions) membrane consisting of a synthetic (PCL) and natural (gelatin) polymer with an optimum mixing ratio of 9.35% PCL and 6.34% gelatin [w/v solvent] for lung application [21,22]. The BETA membrane contains a sacrificial component (gelatin), which provides enough space for the cells to deposit their own ECM during proliferation. In the present study, we exploit this feature of the BETA membrane to improve the biomimicry of the BETA scaffold. We introduce a hybrid bioactive scaffold consisting of a synthetic co-polymeric BETA membrane acting as a backbone of the ECM in the alveolar-capillary tissue, which is then biologically enhanced by cell-derived ECM to closely mimic the microenvironment of the pulmonary cells. The results show that the integrated cell-derived ECM with a supporting biomimetic membrane can not only mimic the composition and 3D structure of the lung ECM, but also enhances the physiologic biomechanical and biophysical properties of the scaffold for LTE application.

BETA Scaffold
The BETA scaffold was manufactured, as previously described [21]. Briefly, the copolymer emulsion of poly(ε-caprolactone) (PCL: Sigma-Aldrich, Saint Louis, MO, USA, Mn 80,000), and gelatin (Type A from porcine skin, Sigma) dissolved in TFE ((2,2,2-trifluoroethanol; Roth) were spin-coated (2000 rpm) and were dried under vacuum (300 mbar). The optimum mixing ratio of 9.35% PCL and 6.34% gelatin [w/v solvent] was used for the co-polymer of PCL/gelatin as previously described [22]. Membranes were sterilized before cell culture experiments with PBS, ethanol 80%, and UV exposure. Scanning electron microscopy (SEM, Zeiss Crossbeam 340, Carl Zeiss AG, Oberkochen, Germany) was used to study the morphology of the BETA scaffolds. The cells were fixed in 2% v/v glutaraldehyde (GA; Sigma-Aldrich) and then dehydrated in gradient acetone solutions (Sigma-Aldrich) followed by hexamethyldisilazane (HDMS, Sigma-Aldrich) for 15 min. The samples were subsequently sputter-coated with platinum and analyzed at an operating voltage of 2 kV.

Chick Chorioallantoic Membrane (CAM) Assay
The ex ovo chick chorioallantoic membrane (CAM) assay was employed to examine the biocompatibility and formation of ECM on the BETA scaffold. Fertilized White Leghorn chicken eggs were purchased from LSL Rhein-Main (Dieburg, Germany). After sterilization of the eggs with 80% EtOH, they were incubated at 37 • C with 60% humidity. The BETA scaffolds were placed on top of the CAM on day 6, and the eggs were resealed with parafilm. The scaffold and the surrounding tissues were excised together on day 10 and fixed with 4% PFA for 1 h at room temperature. The fixed samples were then dehydrated in a 30% w/v (overnight at 4 • C) and 15% w/v (overnight at 4 • C) sucrose solution, and embedded in an optimal cutting temperature compound (OCT Embedding Matrix, Carl Roth, Germany) for sectioning. To evaluate the ECM formation and connective tissue, Masson's trichrome (Carl Roth, Germany) was used according to the standard protocols, and images were acquired using a Zeiss Axio Scan Z.1 slide scanner.

ECM-Integrated BETA Scaffold
IMR90 cells with a cell density of 40,000 cells cm −2 were seeded on a BETA scaffold and grown in a culture medium of DMEM/F12 supplemented with 10% FBS, 1% L-glutamine (2 × 10 −3 M, Gibco), 2-phospho-L-ascorbic acid (0.1 × 10 −3 M, Sigma), and 1% (v/v) Pen/Strep. The cells were also treated with 5 ng mL −1 recombinant human TGFβ1 protein (R&D Systems, Minneapolis, MN, USA) to enhance ECM production. After 5 days, the cells were decellularized at 37 • C, as described in Table 1, based on the literature [24][25][26][27]. After decellularization, the BETA scaffolds were washed three times with PBS. To prevent unwanted DNA, the acellular BETA scaffolds were incubated with 10 U/mL DNAse I, RNase-free (Thermo Scientific EN0525) in PBS with Ca 2+ , and Mg 2+ for 1 h at 37 • C. After decellularization and DNAse treatment, the ECM-integrated BETA scaffolds were used for the repopulation step. Among the decellularization protocols, the optimized method (20 mM NH 4 OH and 0.1% Triton X-100), was performed and the ECM-integrated BETA scaffold was subsequently repopulated with the phLFs with a cell density of 40,000 cells cm −2 in the culture medium of DMEM/F12 supplemented with 10% FBS and 1% (v/v) Pen/Strep for 5 days. The scaffolds were also seeded with the human bronchial epithelial 16HBE14o − cell line with cell density and 150,000 cells cm −2 in the culture medium of MEM (Gibco) supplemented with 10% FBS and 1% v/v Pen/Strep (100 U mL −1 ) for 6 days. The samples were then fixed with 4% paraformaldehyde (PFA; Sigma-Aldrich) for 20 min at room temperature for IF analysis. The WST1 colorimetric metabolic activity assay (Roche, Mannheim, Germany) was used to evaluate the cell viability. Briefly, 1 mL of diluted WST1 reagent (1:10) was incubated with the cells for 20 min and the supernatant (200 µL) was transferred to a 96-well plate. The absorbance was measured at 450 nm (Safire2TM, Tecan, Männedorf, Switzerland).

Functional Analysis
The transepithelial electrical resistance (TEER) measurement (Millicell ERS-2, Millipore, Burlington, MA, USA) was used to assess the barrier integrity in 16HBE14o − cells. To obtain TEER values (Ω cm 2 ), the measured resistance in ohm (Ω) is subtracted from the resistance of the membrane (without cells) and then multiplied by the effective growth surface area (cm 2 ; here 4.2 cm 2 ).
To measure the apparent permeability, FITC-dextran 4 kDa (FD4, Sigma-Aldrich) (1 mL in PBS) was added to the apical side of the membrane (surface area 4.2 cm 2 ). Every 30 min, 100 µL of FD4 in the basal side (total volume 2 mL) was taken in a black 96-well plate and analyzed by the plate reader (Safire2 TM , Tecan; excitation: 483 nm, emission: 525 nm). The apparent permeability coefficient (P app , cm s −1 ) was calculated with Equation (1).
where dQ/dt is the steady-state flux or the transport rate; A is the surface area of the membrane; C 0 is the initial concentration of FITC-dextran 4 kDa (FD4, Sigma-Aldrich) added to the apical compartment (mg mL −1 ).

Results
Here, we developed a bioactive scaffold based on cell-derived ECM, which was integrated with the BETA membrane ( Figure 1). The BETA membrane has already been introduced by us, which is thin (thickness is 5 µm) with high wettability (WCA: 68 ± 5) [21]. The BETA membrane is stretchable with an elastic modulus of 0.78 ± 0.24 MPa and its fatigue test showed no plastic deformation or creep [21]. The BETA has 10% surface porosity and permeability of 8.18 ± 2.54 × 10 −6 cm s −1 for 4 kDa FITC-dextran [21]. The cytocompatibility of the BETA membrane has been evaluated using lung epithelial cell lines such as human lung alveolar epithelial (A549) and human bronchial epithelial (16HBE14o − ) cells, which showed no systematic toxicity or leaching of unwanted materials to the culture medium ( Figure 2a) [21,22].
Biomedicines 2022, 10, x FOR PEER REVIEW 5 of 12 such as human lung alveolar epithelial (A549) and human bronchial epithelial (16HBE14o − ) cells, which showed no systematic toxicity or leaching of unwanted materials to the culture medium ( Figure 2a) [21,22].  Biomedicines 2022, 10, x FOR PEER REVIEW 5 of 12 such as human lung alveolar epithelial (A549) and human bronchial epithelial (16HBE14o − ) cells, which showed no systematic toxicity or leaching of unwanted materials to the culture medium ( Figure 2a) [21,22].   In the present study, we also showed the biocompatibility of the BETA using the chick chorioallantoic membrane (CAM) assay (Figure 2b-d). The analysis of the surrounding tissues/BETA showed the deposition of connective tissue and ECM proteins (such as collagen) on the BETA scaffold (Figure 2c,d). According to the CAM assay, we also found that the cells could deposit their own ECM onto the BETA scaffold (Figure 2c,d). Masson's trichrome analysis showed that fibrous connective tissues are resigning on and within the BETA membrane. To obtain a bioactive scaffold, we first populated the BETA membrane with the IMR90 human pulmonary fibroblast cell lines to deposit the ECM proteins into the scaffold (Figure 3a). It has been shown that transforming growth factor-β1 (TGF-β1) stimulates pulmonary cells to produce matrix proteins [26]. Thus, we stimulated the fibroblasts with TGFβ1 as a fibrotic agent to stimulate the innate production of their own ECM proteins into the BETA membrane. We showed that the treatment of mouse embryonic fibroblasts (MEF) on the BETA membrane with 5 ng mL −1 TGFβ1 upregulates cell proliferation and modulates the fibroblast phenotype, as indicated by Ki67, which is associated with cellular proliferation (Figure 4).
After almost 3 days of cell culture, the gelatin was completely dissolved from the BETA membrane (PCL/gelatin), leaving pores for the expansion of cells and their ECM (Figure 2a, left panel). The IF analysis showed the formation of a confluent layer of activated fibroblasts on the BETA scaffold, characterized by the F-actin cytoskeleton and αSMA (a marker of myofibroblast) (Figure 3a). The populated membrane with fibroblasts were subsequently (gently) decellularized to preserve the key proteins of ECM and improve the bioactivity of the BETA scaffold (Figure 3b). We followed several protocols for decellularization by changing the detergent type, the concentration of detergent, and the decellularization time, which are widely used in the literature [24][25][26][27]. Both NH 4 OH and Triton X-100 are typically considered as mild decellularization agents compared to the invasive sodium dodecyl sulfate (SDS) [24][25][26]28]. The full details of the protocols are provided in Table 1.
The IF analysis of the three main protocols showed that protocol 2 (20 mM NH 4 OH and 0.1% Triton X-100 for 40 min) is a superior protocol in terms of the complete removal of the cells (both cell nuclei, DAPI, and F-actin cytoskeleton) and at the same time, preserving collagen I and IV, the main proteins of the ECM (Figure 3b). To fully remove the DNA materials, which harms the further recellularization step, we incubated the acellular scaffold with DNase I for 1 h. We used this optimized protocol for the recellularization step. To assess the feasibility of the re-population of cells on the decellularized scaffold, we used primary fibroblasts from human (without chronic lung disease (non-CLD) donors) and human bronchial epithelial (16HBE14o − ) cells. The re-populated scaffold with primary fibroblasts derived from non-CLD patients showed the formation of the F-actin cytoskeleton ( Figure 5 top panel) with almost no cytotoxicity effect (WST-1 assay, data not shown). The IF analysis showed the formation of a monolayer of 16HBE14o − as characterized by the F-actin cytoskeleton (Figure 5b bottom panel). The transepithelial electrical resistance (TEER) and apparent permeability of the monolayer of 16HBE14o − were measured as 827 ± 178 Ω cm 2 and 4.2 × 10 −6 cm/s, respectively.
stimulates pulmonary cells to produce matrix proteins [26]. Thus, we stimulated the fi broblasts with TGFβ1 as a fibrotic agent to stimulate the innate production of their own ECM proteins into the BETA membrane. We showed that the treatment of mouse embry onic fibroblasts (MEF) on the BETA membrane with 5 ng mL −1 TGFβ1 upregulates cel proliferation and modulates the fibroblast phenotype, as indicated by Ki67, which is associated with cellular proliferation (Figure 4).   Table 1. Collagen I (magenta), collagen IV (red), F-actin cytoskeleton (green), and cell nuclei (DAPI, blue). Scale bars are 100 µm. removal of cells retaining collagen I and IV as the two key ECM proteins. Protocol 2 (20 mM NH4OH and 0.1% Triton X-100 for 40 min) with DNAse I treatment (100 U/mL in PBS with Ca 2+ and Mg 2+ ) was preferred as the optimized decellularization protocol. Details of the decellularization protocols are shown in Table 1. Collagen I (magenta), collagen IV (red), F-actin cytoskeleton (green), and cell nuclei (DAPI, blue). Scale bars are 100 µm. Figure 4. The IF analysis of mouse embryonic fibroblasts (MEF) on the BETA membrane without and with the treatment with TGFβ1 as a fibrotic agent. Ki67, which is associated with cellular proliferation, is amplified after the induction of MEF with 5 ng mL −1 TGFβ1. Ki67 (magenta), F-actin cytoskeleton (green), nuclei (DAPI, blue). Scale bars are 100 µm.
After almost 3 days of cell culture, the gelatin was completely dissolved from the BETA membrane (PCL/gelatin), leaving pores for the expansion of cells and their ECM (Figure 2a, left panel). The IF analysis showed the formation of a confluent layer of activated fibroblasts on the BETA scaffold, characterized by the F-actin cytoskeleton and αSMA (a marker of myofibroblast) (Figure 3a). The populated membrane with fibroblasts were subsequently (gently) decellularized to preserve the key proteins of ECM and improve the bioactivity of the BETA scaffold (Figure 3b). We followed several protocols for decellularization by changing the detergent type, the concentration of detergent, and the decellularization time, which are widely used in the literature [24][25][26][27]. Both NH4OH and Triton X-100 are typically considered as mild decellularization agents compared to the invasive sodium dodecyl sulfate (SDS) [24][25][26]28]. The full details of the protocols are provided in Table 1.
The IF analysis of the three main protocols showed that protocol 2 (20 mM NH4OH and 0.1% Triton X-100 for 40 min) is a superior protocol in terms of the complete removal of the cells (both cell nuclei, DAPI, and F-actin cytoskeleton) and at the same time, preserving collagen I and IV, the main proteins of the ECM (Figure 3b). To fully remove the DNA materials, which harms the further recellularization step, we incubated the acellular scaffold with DNase I for 1 h. We used this optimized protocol for the recellularization step. To assess the feasibility of the re-population of cells on the decellularized scaffold, we used primary fibroblasts from human (without chronic lung disease (non-CLD) donors) and human bronchial epithelial (16HBE14o − ) cells. The re-populated scaffold with primary fibroblasts derived from non-CLD patients showed the formation of the F-actin cytoskeleton ( Figure 5 top panel) with almost no cytotoxicity effect (WST-1 assay, data not shown). The IF analysis showed the formation of a monolayer of 16HBE14o − as characterized by the F-actin cytoskeleton (Figure 5b bottom panel). The transepithelial electrical resistance (TEER) and apparent permeability of the monolayer of 16HBE14o − were measured as 827 ± 178 Ω cm 2 and 4.2 × 10 −6 cm/s, respectively.

Discussion
Several bottom-up and top-down approaches have been used to engineer lung tissue. Whole or partial organ decellularization is a highly promising method to maintain the main structure and key proteins of the lung, which then facilitates the recellularization of autologous stem or progenitor cells [8,[29][30][31][32]. The application of whole organ decellularization, however, has been limited due to the immunogenicity of the decellularized scaffold during the recellularization process [33,34]. In addition, one of the main challenges of the available approaches toward functional lung tissue is the inadequate alveolar−capillary barrier function in the constructed tissue due to the lack of biomimetic scaffold [29,35].
Here, we introduced a hybrid bioactive scaffold to closely mimic the microenvironment of the alveolar-capillary barrier. The hybrid bioactive scaffold consisted of (i) BETA scaffold and (ii) ECM-derived cells. In the co-polymeric BETA scaffold of PCL/gelatin, we used PCL, which is widely used in TE and regenerative medicine applications, mainly

Discussion
Several bottom-up and top-down approaches have been used to engineer lung tissue. Whole or partial organ decellularization is a highly promising method to maintain the main structure and key proteins of the lung, which then facilitates the recellularization of autologous stem or progenitor cells [8,[29][30][31][32]. The application of whole organ decellularization, however, has been limited due to the immunogenicity of the decellularized scaffold during the recellularization process [33,34]. In addition, one of the main challenges of the available approaches toward functional lung tissue is the inadequate alveolar−capillary barrier function in the constructed tissue due to the lack of biomimetic scaffold [29,35].
Here, we introduced a hybrid bioactive scaffold to closely mimic the microenvironment of the alveolar-capillary barrier. The hybrid bioactive scaffold consisted of (i) BETA scaffold and (ii) ECM-derived cells. In the co-polymeric BETA scaffold of PCL/gelatin, we used PCL, which is widely used in TE and regenerative medicine applications, mainly due to its biocompatibility, mechanical properties, and suitability for modification. The application of PCL for the pulmonary cells has been shown by us [21,22] and other groups [11,14]. In the BETA scaffold, we also adopted gelatin as a delayed dissoluble porogen to (i) promote initial cell attachment by integrin-mediated interactions via its cell-responsive motifs such as arginine-glycine-aspartic acid (RGD) sequence, and (ii) provide a space for cell infiltration after (gelatin) removal, leaving a cavity. Gelatin is a bioresorbable and reproducible polymer with tunable biophysical properties and very low immunogenicity compared to native ECM polymers such as collagen [16,36]. The PCL/gelatin membrane has been designed to recapitulate the main physico-mechanical characteristics of the alveolar-capillary barrierthe gas exchange area in the lung. The BETA scaffold is also self-supported in the absence of any toxic cross-linker.
We have already shown that BETA has connective porosity by using focused ion beam-scanning electron microscopy (FIB-SEM) tomography, which allows the cells to infiltrate and reside in the scaffold, interact with other cells, and form their own ECM materials [21]. We exploited this advantage of the BETA membrane to make it even more biomimetic through the deposition of the natural ECM proteins by pulmonary cells (Figure 1). The deposition of cell-derived ECM onto synthetic scaffolds is a promising approach to mimicking the microenvironment and natural complexity of the region of interest with favorable biological characteristics [37]. Among the human lung fibroblasts, IMR90 (a fibroblast isolated from the normal lung tissue) has shown a great potential to produce a high density of cell-derived ECM [27]. We therefore employed IMR90 as a sacrificial ECM generator for the biomimetic scaffold. We stimulated the fibroblasts with a profibrotic agent of TGFβ1 to differentiate them from myofibroblasts (characterized by αSMA marker, see Figure 3a). The activated fibroblasts (myofibroblasts) can produce high amounts of ECM proteins in response to pro-fibrotic stimuli [38].
The analysis of the barrier integrity dynamics, measured by TEER and apparent permeability, showed that the monolayer of 16HBE14o − cells on the bioactive BETA scaffold could form a tighter barrier compared to the established models on Transwell inserts. The TEER value obtained for 16HBE14o − was almost 2-times higher than the already reported data in the literature [39,40]. The apparat permeability also showed a firmer paracellular barrier against FITC-dextran (4 kDa), which is in line with the reported value in the literature with a range of 1.5-4 × 10 −6 cm/s [39,40].
The collagens (types I, III, and IV) and elastin, fibronectin, and glycosaminoglycans (GAGs) are the main components of the ECM, which play a key role in the proliferation, differentiation, and migration of pulmonary cells during physiological and non-physiological (disease) conditions [38]. The deposited ECM scaffold showed an excellent regeneration of the linear ECM proteins (such as collagen I), indicating the high degree of physiologic relevance and ECM microenvironment in the lung region, in particular, the linear pattern of the ECM during disease, which is not obtained by solubilized ECM such as Matrigel or hydrogels [27]. The ECM-derived scaffolds shown enhanced cell infiltration and favorable immune responses for the cell hemostasis upon in vivo implantation [41].
Here, we developed a bioactive scaffold based on the ECM-derived human cells. It has been shown that human-based cell models can improve the predictivity of the in vitro models for drug development [42]. This feature is one of the main advantages of our ECM-derived scaffold over the already developed ones based on the decellularized animal models [8,31]. Using this method, the unique niches of the region of interest can be explored, for example, a specific disease can be mimicked due to the formation of the native ECM proteins, which can subsequently guide the cells during proliferation, migration, and infiltration. The supporting BETA membrane mimics the pore size and elastic modulus of the basement membrane in the alveolar-capillary region [21], which enhances the functionality of the regenerated tissue. This is very important since obtaining a connective porous structure is not easily accessible via only ECM-derived or naturalbased scaffolds.
In summary, our bioactive scaffold benefits from (i) the biomimetic microenvironments for the cells due to the cell-deposited ECM, and (ii) an interconnected porosity for migration and special reorganization of the cells, thanks to the supporting BETA membrane. The bioactive BETA scaffold presented excellent bioactivity for pulmonary epithelial cells (16HBE14o − ) and primary fibroblasts that were derived from non-CLD patients. The pulmonary epithelial cells could also form a tight barrier integrity, which is one of the main challenges in engineered lung tissue.

Outlook
The BETA membrane has already been shown its ability for particokinetic and pharmacokinetic studies in the lung [22]. In the present study, we extended its potential application for LTE as an allograft or xenograft transplantation by improving its biomimicry. We used a human fibroblast cell line to deposit ECM onto a synthetic BETA membrane. However, fibroblasts derived from patients with idiopathic pulmonary fibrosis (IPF) can also be employed to mimic the pathological pattern of the ECM during disease conditions such as fibrosis with higher stiffness. Moreover, the introduced cell-derived ECM scaffold is tunable and can be adjusted for other specific-tissue applications.


s2fieldsofstudy: Biology Psychology Medicine

text:
Inflammatory pathophysiological mechanisms implicated in postpartum depression

Postpartum Depression (PPD) is a serious psychiatric disorder of women within the first year after delivery. It grievously damages women’s physical and mental health. Inflammatory reaction theory is well-established in depression, and also has been reported associated with PPD. This review summarized the inflammatory pathophysiological mechanisms implicated in PPD, including decreased T cell activation, increased proinflammatory cytokines secretion, active kynurenine pathway, and initiated NLRP3 inflammasome. Clinical and preclinical research are both gathered. Potential therapeutical alternatives targeting the inflammatory mechanisms of PPD were introduced. In addition, this review briefly discussed the differences of inflammatory mechanisms between PPD and depression. The research of inflammation in PPD is limited and seems just embarking, which indicates the direction we can further study. As a variety of risky factors contribute to PPD collectively, therapy for women with PPD should be comprehensive, and clinical heterogeneity should be taken into consideration. As PPD has a predictability, early clinical screening and interventions are also needed. This review aims to help readers better understand the inflammatory pathological mechanisms in PPD, so as to identify biomarkers and potential therapeutic targets in the future.

Introduction
Postpartum Depression (PPD) is defined as the onset of major depressive disorder (MDD) of women within the first year after delivery (Mughal et al., 2022). It is a serious psychiatric disorder and grievously damages women's physical and mental health (Balaram and Marwaha, 2022). The clinical symptoms include gloomy mood, reduced interest or pleasure in matters, weariness, insomnia, inappropriate guilt, excessive concern or indifference to infants, and even suicide (Raza and Raza, 2022). The prevalence of PPD is about 15% all over the world (Mughal et al., 2022). Due to the diverse economic levels and screening awareness, the incidence of PPD is different in various regions and may be underestimated (Shorey et al., 2018). PPD not only affects women's emotion and cognition, but also damages the mother-infant relationship and the growth of young children (Badr et al., 2018;Slomian et al., 2019). It has been reported that child whose mother suffers from PPD is more likely to suffer from depression (Abdollahi et al., 2017; OPEN ACCESS EDITED BY Tao Xu, Anhui Medical University, China Weissman, 2018;Tainaka et al., 2022). PPD also breaks family harmony and has become a serious social problem (Letourneau et al., 2012). The pathological mechanism of PPD is multifactorial and has not been fully clarified. The potential pathogenesises are as follows ( Figure 1): dysfunction of hypothalamic-pituitary-adrenal (HPA) axis, imbalance of hormones (estradiol, progesterone, oxytocin, corticotropin releasing hormone, etc.), imbalance of neurotransmitters (serotonin, dopamine, γ-aminobutyric acid, glutamate, etc.), imbalance of neurosteroids (allopregnanolone, etc.), epigenetics, alterant synaptic connection, neuroinflammation, and so on (Payne and Maguire, 2019;Stewart and Vigod, 2019;Mughal et al., 2022). The therapeutic modalities of PPD mainly include psychotherapy and medical treatment, which are similar with the treatment of conventional depression (Brummelte and Galea, 2016;Stewart and Vigod, 2019). It is worth noting that patients with PPD, who may be breastfeeding, should avoid medications that affect the infants (Becker et al., 2016). Exposure to antidepressants in late pregnancy could lead to neonatal adaptation disorders, such as drowsiness and irritability. According to the available evidence, sertraline and amitriptyline are the preferred antidepressants (Wisner et al., 1996;Hantsoo et al., 2014;Cuomo et al., 2018). Brexanolone, a positive allosteric modulator of γ-aminobutyric acid (GABA) A receptors, is approved as the first drug expressly for treating women with PPD (Kanes et al., 2017;Gunduz-Bruce et al., 2022).
Frontiers in Pharmacology frontiersin.org It further impairs the integrity of the BBB (Haruwaka et al., 2019). Thus, a positive circuit of inflammatory response is generated, which aggravates the nerve injury (Linnerbauer et al., 2020). Furthermore, the proinflammatory cytokines activate the HPA axis, which in turn increases the production of cortisol (Noushad et al., 2021). The tryptophan (Trp)-kynurenine (Kyn) pathway is activated as well. Subsequently, the synthesis of quinolinic acid and 3-hydroxykynurenine is increased, which induces oxidative stress and nerve injury (Tattersfield et al., 2004;Mackay et al., 2006). Neuroinflammation is also associated with a variety of neurodegenerative diseases, such as Parkinson's disease (Tansey et al., 2022), Alzheimer's disease (Leng and Edison, 2021) and so on (Fontana et al., 2021). PPD is also reported to be closely related to neuroinflammation. In this review, we focused on the potential inflammatory mechanisms to underpin PPD pathophysiology.

Inflammatory pathophysiological mechanisms in postpartum depression
Inflammatory responses can occur in the periphery or central nervous system. Pro-inflammatory and antiinflammatory responses are the two types of inflammatory reactions. Pregnancy is linked to specific immunological Treg cells ↓ hormone-simulated pregnancy induced PPD rat model immune suppression Qu et al. (2015) women with PPD DNA methylation in CD3↑ Robakis et al. (2020) women with PPD Treg cells ↓ Weigelt et al. (2013) women with PPD Treg cells ↓ 3-hydroxyanthranilic acid↑ hormone-simulated pregnancy induced PPD mouse model NLRP3 inflammasome↑ Zhu and Tang, (2020) hormone-simulated pregnancy induced PPD rat model NLRP3 inflammasome↑ Abdul Aziz et al. (2021) hormone-simulated pregnancy induced PPD rat model NLRP3 inflammasome↑ Zhai et al. (2022) responses that protect the fetus from the mother immune system. In order to support immunosuppression, antiinflammatory cytokines are increased, while proinflammatory cytokines are decreased during pregnancy (Al-Azemi et al., 2017;Kwiatek et al., 2021). In response to the physical damage and exertion associated with labor, the antiinflammatory milieu transforms to a pro-inflammatory state after delivery (Miyoshi et al., 2021). In this section, we will review evidence of inflammatory pathophysiological mechanisms in PPD (Table 1), including roles of T cells, cytokines, kynurenine and inflammasomes.

T cells
T cells are essential for the control and clearance of most infections. Major histocompatibility complex (MHC) proteins present short peptide antigens to T cell receptors, and T cells respond to infections in such an antigen-specific way (Kumar, 2018). It plays a key role in adaptive immunity by mediating helper functions to the immune system of the body (Dong, 2021). In healthy women, the postpartum period is a time of increased T cell activation (Osborne et al., 2020). Women with PPD do not have physiologically increased T-cell activity after giving birth. Lauren M Osborne et al. (Osborne et al., 2020) found that T cells were significantly higher in postpartum women without PPD than in healthy non-postpartum controls. Increases of TH1 cells and T regulatory (Treg) cells drove the immunological enhancement in healthy postpartum women, which were absent or muted in women with PPD (Osborne et al., 2020). Similar results were obtained in animal experiments. It was reported that immune suppression occurs 2 weeks after hormone withdrawal in hormone-simulated pregnancy induced PPD rat model (Qu et al., 2015). At present, there are few reports on the possible mechanism of the shift of immune state (from immune suppression to immune activation) before and after childbirth. It was speculated that changes in DNA methylation density in CD3 may be associated to depression during pregnancy (Robakis et al., 2020). Another research implied that lower microRNA-146a expression in monocytes was linked to lower natural Treg cells in PPD (Weigelt et al., 2013). Daniela Krause et al. (2014) declared that Treg cells are reduced both antepartum and postpartum in women with PPD, and the level of Treg cells in pregnancy might be a forecast for PPD. In conclusion, there is much evidence that PPD is accompanied by decreased T cell activation. The compensation of the monocytic system could be a probable result of the T cellsmediated immunosuppression in depressive women (Krause et al., 2014). Monocytes that may pass the BBB, appear to be important in the pathophysiology of depression as contributing to an inflammatory environment in the brain and leading nerve scathe.

Cytokines
Cytokines include pro-inflammatory and anti-inflammatory types. Proinflammatory cytokines could access the brain through the BBB and participate in many pathophysiologic processes including glial cells activation, neurotransmitter metabolism, and so on (Miller and Raison, 2016;Shi et al., 2022). Among the multiple cytokines, interleukin (IL) -6 has been reported most related to PPD. However, the conclusions are somewhat conflicting. The mainstream view is that serum level of IL-6 in women with PPD is increased, compared to healthy puerperal women (Liu et al., 2016;Payne and Maguire, 2019;Achtyes et al., 2020;Nazzari et al., 2020;Sha et al., 2022;Worthen and Beurel, 2022). Other studies (Ahn and Corwin, 2015;Nagayasu et al., 2021) did not find the correlation between IL-6 levels and the scores of Edinburgh Postpartum Depression Scale (EPDS), depressive symptoms, or stress variables. In an exploratory study among postpartum veterans (Szpunar et al., 2021), the researchers found that elevated IL-1β and tumor necrosis factorα (TNF-α) might have a positive correlation with the severity of depressive symptom. And the high level of IL-1β was also related to suicidal thoughts during pregnancy (Szpunar et al., 2021). Similarly, other studies showed that uric or plasmic IL-1β was increased in mothers with depressive symptoms or high scores of EPDS (≥13) (Corwin et al., 2008;Sha et al., 2022). In contrast, R Buglione-Corbett's laboratory (Buglione-Corbett et al., 2018) clarified that serum TNF-α was negatively correlated with EPDS score, and there was no statistically significant associations between depressive symptoms and IL-6 or IL-1β. Serumal interferon-γ (IFN-γ) and the ratio of IFN-γ/IL-10 were decreased in PPD, according to Maureen W Groer et al. (Groer and Morgan, 2007). Besides, the secretion of IL-8 has been reported to increase in the postpartum period (Szpunar et al., 2021). Increased plasma IL-8 or reduced IL-2 was associated with higher risk for PPD (Achtyes et al., 2020). Chemokine is a small molecule cytokine capable of chemotactic cell directional movement. Chemokines and their receptors mediate cell migration, thereby affecting a variety of basic biological processes and disease conditions, such as inflammation and cancer. C-X-C motif chemokine 1 (CXCL1) was reported to significantly elevate in women with PPD (Brann et al., 2020). In general, levels of many cytokines alter in the postpartum period and might potentially become inflammatory biomarkers for PPD.

Kynurenine
Increased inflammation raises the production of the broadly distributed enzyme indoleamine 2,3-dioxygenase (IDO) (Cervenka et al., 2017). Activation of the HPA axis promotes the hepatic enzyme tryptophan 2,3-dioxygenase (TDO). Both enzymes transform tryptophan (Trp) into kynurenine (Kyn), Frontiers in Pharmacology frontiersin.org 04 which is then converted into downstream neurotoxic metabolites, i. e, quinolinic acid and kynurenic acid, to damage neurons (Savitz, 2020). On the other hand, Trp is the precursor of neurotransmitter serotonin (5-hydroxytryptamine, 5-HT). The increased conversion of Trp to Kyn results in less synthesis of 5-HT, which leads to depressive symptoms (Cervenka et al., 2017). Chengxuan Quan et al. (2020) showed that women with PDD had significantly greater Kyn levels 1 day before delivery compared to the control group. Women with PDD had significantly lower kynurenic acid level, higher quinolinic acid level, and higher quinolinic acid/kynurenic acid ratio 3 days after delivery than women without PPD. Similarly, a study  showed that women with PPD had significantly higher serum Kyn and quinolinic acid concentrations, and lower serum kynurenic acid concentrations 3 days after cesarean section. Qiong Sha et al. (2021) declared that estrogen and progesterone were respectively negatively correlated with Kyn and quinolinic acid in the postpartum period. Animal research (Qiu et al., 2021) also showed that postpartum corticosterone could influence Trp-Kyn pathway, inducing the production of neurotoxic metabolites 3-hydroxykynurenine and 3-hydroxyanthranilic acid. On the contrary, Eric Achtyes et al. (2020) discovered that down-regulation of quinolinic acid was related to high risk for PPD. In general, the activation of Kyn pathway is implicated in PPD as evidenced by many studies (Nazzari et al., 2020). The changes of metabolites of Kyn in postpartum are still conflicting and need to be further researched.

Inflammasomes
The Nod-like receptor protein (NLRP) inflammasomes are protein complexs that exert important roles in neuroinflammation. Among the various inflammasomes, NLRP3 inflammasome is the most studied one (Huang et al., 2021). When stimulating by risky factors such as adenosine triphosphate, the adapter molecule apoptosis-related speck-like protein (ASC) and pro-caspase-1 are recruited by NLRP3 and form a protein complex . Pro-caspase-1 is then transformed into mature caspase-1. Whereafter, caspase-1 mediates the maturation of the proinflammatory cytokines IL-1β and IL-18. The secretion of proinflammatory cytokines leads to downstream inflammatory cascade and cell pyroptotic death (Zhu et al., 2018;Huang et al., 2021). Jialei Zhu et al. (Zhu and Tang, 2020) firstly proposed that astrocytic NLRP3 inflammasome was activated in the hippocampus of PPD mouse model. Another study (Abdul Aziz et al., 2021) also showed that increased NF-lB/NLRP3/caspase-1 activity was detected in the hippocampus of PPD rat model. Similarly, a recent study (Zhai et al., 2022) clarified that NLRP3 inflammasome was activated in the hypothalamus of PPD rat model. Although there have been many reports on the correlation between inflammasomes and the pathological mechanism of depression, the research of inflammasomes in PPD is just embarking. Thus far, there is no study of inflammasome in patients with PPD, and there are only a few animal experiments using hormone-simulated pregnancy induced PPD model. It deserves further study.
3 Potential therapeutical alternatives for postpartum depression targeting the inflammatory mechanisms At present, the only approved drug specifically used for the treatment of PPD is brexanolone. It is soluble allopregnanolone and targets GABAergic system. It has been reported that injection of allopregnanolone reduced microglial activation and astrocyte proliferation in mouse model (Liao et al., 2009). Another study showed that allopregnanolone synthesis was reduced by IL-6 (Parks et al., 2020). It indicates the potential significance of anti-inflammatory therapy for PPD. In this section, we will review potential therapeutical alternatives for PPD targeting T cells, cytokines, kynurenine or NLRP3 inflammasome in clinical trials.

T cell-based immunotherapy
T cell-based immunotherapy has received great attention in tumor treatments (Zhang, K. et al., 2022) in recent years. Engineering T cells is a rapidly advancing technology and is a good strategy for stimulating T cells proliferation to effectively target tumors (Belk et al., 2022). However, it may induces serious adverse effects, such as nonspecific inflammation (Belk et al., 2022). Chimeric-antigen receptor (CAR) T cells, as the first commercial products, are approved for hematologic malignancies (Greenbaum et al., 2021). So far, there has been none engineering T cells applicated in the treatment of depression. And it seems be "making a mountain out of a molehill". The supplementation of some substances regulating T cells in diet or drugs may be better for PPD. In a prospective, randomizedcontrolled study, trace element selenium (Se) was found to upregulate the activated Treg cells . Naghmeh Mokhber et al. (2011) conducted a trial to determine the impact of prenatal Se supplementation on women's levels of PPD. Primigravid pregnant women were randomly assigned to receive Se or placebo every day up until birth. The mean EPDS score in the Se group was markedly lower than that of the control group. It suggests that supplementation with Se during pregnancy would be an effective strategy for the prevention of PPD. Traditional Chinese Medicine and its extracts are also reported to have immunity-enhancing capacity . Leonurus has the effect of regulating menstruation and plays an auxiliary role in the treatment of gynecological diseases. Leonurine, Frontiers in Pharmacology frontiersin.org the extract of Leonurus, was found to regulate Treg/Th17 balance (Du et al., 2020). It exerted antidepressant effects in chronic mild stress-induced depression mouse model (Jia et al., 2017).

Cytokine inhibitors
Cytokine inhibitors are commonly used clinically in autoimmune diseases such as rheumatoid arthritis (RA). The role of cytokine inhibitors in depression is still controversial. Tocilizumab is a recombinant humanized anti-human IL-6 receptor monoclonal antibody. A study demonstrated a favourable impact of tocilizumab therapy on anxiety and depression in patients with RA (Tiosano et al., 2020). However, another study showed that blockade of the IL-6 receptor with tocilizumab resulted in significantly more depressive symptoms (Knight et al., 2021). Anti-TNF-α compounds were reported as a potential therapeutic strategy for depression (Uzzan and Azab, 2021). In a randomized controlled trial, TNF antagonism infliximab improved depressive symptoms in patients with high baseline inflammatory biomarkers (Raison et al., 2013). IL-1 receptor antagonist (IL-1RA) is a specific competitive inhibitor of IL-1. It binds to IL-1R and blocks the binding of IL-1α/IL-1β with IL-1R (Maes et al., 2012). A recent study in mouse model demonstrated that the blockade of IL-1R/NF-κB pathway reduced the secretion of complement C3 from astrocyte and regulated synaptic pruning in the prefrontal cortex of depression (Zhang, M.M. et al., 2022). However, a case report showed that IL-1RA anakinra induced depression (Jonville-Bera et al., 2011), which was firstly found to be a new side effect of anakinra. C-X-C motif chemokine receptor 2 (CXCR2) is the receptor of chemokine CXCL1, and the inhibitor of CXCR2 (SB265610) prevented chronic stressinduced depression-like behaviors in mice (Chai et al., 2019). However, there has been no relevant clinical studies. The effect of cytokine inhibitors on depression is mostly carried out in patients with inflammatory diseases (Beurel et al., 2020). It is yet unclear if the improvement is caused, at least in part, by cytokine inhibitor methods' effects on somatic disorders, but from all of these data, depressed individuals with prominent inflammation benefits from them.

IDO and TDO inhibitors
One potential strategy for treating depression is to directly target kynurenine synthesis and reduce its harmful downstream metabolites. Therefore, the straightforward process is to suppress IDO and TDO activity in order to stop the accumulation of kynurenine metabolites. The IDO antagonist 1methyltryptophan (1-MT) has been reported to prevent depressive-like behaviors in many animal experiments (O'Connor et al., 2009;Souza et al., 2017). Clinical trials using 1-MT also have been initiated (Lob et al., 2009). TDO inhibitors include allopurinol, nicotinamide and so on (Badawy, 2019). It has been reported that continued use of low-dose allopurinol was associated with a decreased rate of incident depression (Kessing et al., 2019). The possible pro-longevity effects of nicotinamide adenine dinucleotide precursors have caused further growth of nicotinamide consumption as a dietary supplement (Hwang and Song, 2020). In a randomized, double-blind, and placebocontrolled study, nicotinamide-containing supplements loading between meals in quite low dose can improve depressed mood in young adults with subclinical depression (Tsujita et al., 2019). However, there are potential risks for epigenetic alterations associated with chronic use of nicotinamide at high doses (Hwang and Song, 2020). The possible adverse reactions and their mechanisms are not yet clear, which reminds us to use it cautiously.

NLRP3 inflammasome inhibitors
In recent years, NLRP3 inflammasome selective inhibitors are under development.
Most attempts to inhibit NLRP3 inflammasome focus on compounds that directly bind to NLRP3 and inhibit the assembly of NLRP3 inflammasome complex. MCC950 is a small molecular inhibitor of NLRP3 inflammasome and is reported to exert an antidepressive role in animal experiments Liu et al., 2022). At present, there is no clinical trial of MCC950 on depression. OLT1177 is an orally active β-sulfonyl nitrile molecule developed for osteoarthritis, acute gout and heart failure (Marchetti et al., 2018;Aliaga et al., 2021). CY-09 is also an inhibitor of NLRP3 inflammasome potentially used for osteoarthritis, cryopyrin-associated autoinflammatory syndrome (CAPS) and type 2 diabetes (Jiang et al., 2017;. In addition, INF39  and JC-124 (Yin et al., 2018) are inhibitors of NLRP3 inflammasome as well. So far, researchers have not taken OLT1177, CY-09, INF39, and JC-124 into the researches of depression. On the other hand, some medicines have been found to play an antidepressant role by inhibiting NLRP3 inflammasome. A prospective clinical study reveals that pioglitazone metformin complex alleviates psychological distress via inhibiting NLRP3 inflammasome in patients with polycystic ovary syndrome comorbid psychological distress (Guo et al., 2020). Another research identified fluoxetine as a direct NLRP3 inhibitor as it inhibited activation of the NLRP3-ASC inflammasome and inflammatory cytokine release (Ambati et al., 2021).

Discussion
As a subtype of depression with a "special period" (puerperium) and "special population" (delivery women), the Frontiers in Pharmacology frontiersin.org inflammatory mechanisms of PPD are generally overlaps with that in depression. Meanwhile, some differences exist. In depression, Th17 cells are reported accumulated and the Th17/Treg cell balance was dysregulated (Cui et al., 2021). Similar report has been declared in the study of depression and anxiety during pregnancy (Osborne et al., 2019b). However, this has not been reported in PPD. In a metaanalysis studying inflammatory markers in depression (collecting 5166 patients with depression and 5083 healthy controls) (Osimo et al., 2020), the researchers found that IL-6, TNF-α, IL-12, IL-3, IL-18, and sIL-2R were elevated in depression group. The cytokines upregulated in depression are not exactly same as those in PPD. In terms of inflammasomes, besides NLRP3 inflammasome, depression has also been reported to be associated with the activations of NLRP1 , NLRP2 , and AIM2 (Li, Y.K. et al., 2021). In addition to the inflammatory mechanisms mentioned above, recent reports have also shown that depression is related to caspase-gasdermin D-mediated inflammatory programmed cell death, namely pyroptosis (Chai et al., 2022;Li, S. et al., 2021;Yang et al., 2020). It is unknown whether pyroposis also exists in PPD at present, and it is worth exploring in the future. Besides, much evidence suggests an impact of toll-like receptor 4 (TLR4) signaling on depression (Guo et al., 2019;Xu et al., 2020) while it is rarely reported in PPD. On the other hand, microglial M1/M2 polarization plays important roles in mediating the balance between activation and suppression in inflammation (Nakagawa and Chiba, 2014). Many studies have demonstrated that M1 (pro-inflammatory) polarization was related to depression (Kalkman and Feuerbach, 2016;. It needs more researches to explore whether these pathomechanisms are also relevant to PPD. Although many antidepressant agents or methods are not specially used for restraining inflammation, they actually play anti-inflammatory roles. In addition to the medicines mentioned in the previous section, some potential agents have also been reported to exert roles through other anti-inflammatory mechanisms. Isoliquiritin , pinocembrin , pilose antler peptide (Hu et al., 2022), quercetin , etc. ameliorated depression by suppressing pyroptosis in animal models. Arctigenin , safflower extract , baicalin (Guo et al., 2019), Xiao-Chai-Hu-Tang (Shao et al., 2021), puerarin , etc. alleviated depression through TLR4 signaling pathways. Ketamine (Beckett and Niklison-Chirou, 2022;Wu et al., 2022), magnolol (Tao et al., 2021), astragalin , etc. were reported to attenuate depression and produce anti-inflammatory effects by regulating M2 polarization of microglia. The roles of these agents above in PPD need further animal and clinical trials to explore. Besides, non-steroidal anti-inflammatory drugs (NSAIDs) inhibit the synthesis of prostaglandins in the central nervous system and are commonly used clinically for antipyretic, analgesic, anti-inflammatory and anti-rheumatic effects. In the treatment of conventional depression, antiinflammatory agents have shown better effects compared to placebo in several randomized controlled trials (Muller et al., 2006;Mohammadinejad et al., 2015;Alamdarsaravi et al., 2017). However, this view is still controversial (Berk et al., 2020;Husain et al., 2020;Baune et al., 2021). More clinical trials and evidence need to confirm its effect. In addition to the agents, some methods may also have an antidepressant effect by antiinflammation. Acupuncture may achieve treatment effects on depression through suppression of vagal nerve inflammatory responses . Physical exercise can reduce both depression and inflammation (Paolucci et al., 2018). In addition, microbiome-gut-brain axis shows correlation to depression (Carlessi et al., 2021;Donoso et al., 2022). Though recent systematic reviews (Desai et al., 2021;Trifkovic et al., 2022) demonstrated that there was limited evidence about the effectiveness of probiotics on PPD, probiotics is a promising therapeutical alternative. Correct strain selection should be taken into consideration. And further well-designed, robust clinical trials are needed. All the agents and methods (Table 2) provide new therapeutic ideas for treating PPD.
During the postpartum period, many women suffer from obesity, sleep deprivation, mastitis, or diabetes, and so on. There is plenty of evidence that these factors have a high risk of inflammation (Pyorala, 2003;Halim and Halim, 2019;Irwin, 2019;Atrooz and Salim, 2020;Berbudi et al., 2020;Miao et al., 2022;Rohm et al., 2022;Shangraw and McFadden, 2022). Raising infants may be physically and financially stressful for women. It has been reported that stress could induce immune dysfunction and is associated with inflammation (Glaser and Kiecolt-Glaser, 2005;Umamaheswaran et al., 2018). In addition, the hormone levels of women change after childbirth. Estrogen (Kovats, 2015;Xu et al., 2016), progesterone (Patel et al., 2017), oxytocin (Tang et al., 2019;Szeto et al., 2020), and corticotropin releasing hormone (Webster et al., 1998;Nakade et al., 2021) all have been reported related to inflammation. Therefore, the potential mechanisms of PPD are highly interrelated. A variety of risky factors contribute to PPD collectively. Therapy for women with PPD will be multifaceted and comprehensive.
On the other hand, retrospective reports and case registry studies indicates significant degrees of consistency in depression throughout pregnancy to postpartum as well as across several years pre-conception to postpartum (Hipwell et al., 2022). It reminds us that the pathophysiological mechanisms implicated in PPD started on (or even before) the pregnancy, and PPD should be considered within a lifespan perspective. It is the successive process, and early clinical screening and interventions are necessary. With existing technology and clinical knowledge, it might be possible to identify a population at risk of getting PPD (Cellini et al., 2022). Plenty of evidence indicates that multiple factors (Figure 1) including genetics (eg. nearly 50% of heritability), epigenetics (eg., DNA Frontiers in Pharmacology frontiersin.org 07 methylation at the oxytocin receptor gene), neuroactive molecules (eg., lower levels of allopregnanolone during the second trimester, higher levels of β-endorphin at 25 weeks' gestation, higher levels of cortisol at day 14 postpartum, higher levels of corticotropin-releasing hormone during pregnancy, lower levels of oxytocin during the third trimester, hypractive thyroid function at delivery, higher levels of inflammatory markers prenatally and at delivery), psychiatric history (antenatal major depressive disorder, anxiety, or other psychiatric disorder), adverse life events (eg., physical, psychological, or sexual abuse), demographic information (eg., younger or older maternal age, black or hispanic race, low socioeconomic status), and obstetrical outcomes (eg. preterm birth), are potential predictors for PPD (Yim et al., 2010;Sylven et al., 2013;Corwin et al., 2015;Guintivano et al., 2018a;Guintivano et al., 2018b;Osborne et al., 2019a;Bauer et al., 2019;Cao and Wei, 2020;Cevik and Alan, 2021;Grippi, 2021;Lapato et al., 2021;Nelson et al., 2022). In terms of inflammatory Physical exercise reduce secretion of proinflammatory cytokines Paolucci et al. (2018) Probiotics regulate microbiome-gut-brain axis (Desai et al., 2021;Trifkovic et al., 2022) PPD, postpartum depression; Treg cells, regulatory T cells; Th17, T helper cell 17; IL, interleukin; TNF-α, tumor necrosis factor-α; CXCR2, C-X-C motif chemokine receptor 2; 1-MT, 1methyltryptophan; IDO, indoleamine 2,3-dioxygenase; TDO, tryptophan 2,3-dioxygenase; NLRP3, nod-like receptor protein 3; NSAIDs, non-steroidal anti-inflammatory drugs; TLR4, toll-like receptor 4 Frontiers in Pharmacology frontiersin.org 08 markers, increase of Treg cells prenatally (Krause et al., 2014), upregulation of IL-6 and high-sensitivity C-reactive protein (Hs-CRP) at delivery (Liu et al., 2016), high DNA methylation at FOXP3 Treg-cell-specific demethylated region (TSDR) prenatally (Sluiter et al., 2020), increase of the IL-8/IL-10 ratio during the third trimester  are highly correlated with the occurrence of PPD. In addition, a study has demonstrated that the sum of quinolinic acid, Kyn, 3-OHkynurenine and 3-OH-anthranilic acid during pregnancy was closely associated with body image dissatisfaction (Roomruangwong et al., 2018). Furthermore, a recent crosssectional study indicated that maternal and paternal depression were positively associated and served as predictors of one another in the early postnatal period (Zheng et al., 2022). It reminds us that early screening and evaluation (including the partner) is meaningful. In recent years, artificial intelligence is developing rapidly, providing novel methods for perinatal health prediction modeling, diagnostics, early identification, and monitoring (Ramakrishnan et al., 2021). It is hoped that more scientific research and advanced technology will benefit women with PPD in the future.
Collectively, this review summarizes the inflammatory mechanisms implicated in PPD, including decreased T cell activation, up-regulation of proinflammatory cytokines, activation of kynurenine pathway, and activation of NLRP3 inflammasome. The hypothesis diagram and predicted inflammatory markers are shown in Figure 2. At present, some reviews (Payne and Maguire, 2019;Worthen and Beurel, 2022) have reported the roles of inflammation in PPD, but none of them mentioned the effects of inflammasomes. We have further expanded the contents on this basis. There are also some limitations in this review. 1) Some reports have conflicting conclusions, which makes it difficult for us to draw a definite conclusion in the summary. It may be caused by the clinical heterogeneities, including the differences in situations of subjects (race, age, etc.), mode of production (spontaneous delivery, caesarean section, etc.), scoring method (depressive symptom, PPD scale, etc.), the time collecting samples (24 h, 3 days, 3 months, 6 months, etc. after delivery), sampling content (whole blood, serum, plasma, urine, etc.). 2) Overall, there are limited reports about inflammation in PPD. Many experiments have only animal data rather than human data. It may be due to the vulnerability of postpartum population. Further studies are needed. Altogether, this review declares that inflammatory mechanisms play important roles in the pathology of PPD. Furthermore, the inflammatory indicators should be considered possible clinical markers and therapeutic targets in PPD.

Author contributions
JZ wrote the manuscript. JJ corrected the writing. JT conceptualized and supervised the work, and revised the manuscript.

Funding
This work was supported by the grants from National Natural Science Foundation of China (No. 82104148),

FIGURE 2
The inflammatory mechanisms implicated in postpartum depression and potential predicted markers. The inflammatory mechanisms implicated in postpartum depression include decreased T cell activation, up-regulation of proinflammatory cytokines, activation of kynurenine pathway, and activation of NLRP3 inflammasome. The potential predicted inflammatory markers include increased Treg cells prenatally, upregulation of IL-6 and Hs-CRP at delivery, high DNA methylation at FOXP3 TSDR prenatally, increased IL-8/IL-10 ratio during the third trimester, and high level of quinolinic acid, Kyn, 3-OH-kynurenine and 3-OH-anthranilic acid during pregnancy. Trp, tryptophan; Kyn, kynurenine; Treg cells, regulatory T cells; IL, interleukin; Hs-CRP, high-sensitivity C-reactive protein; DNA, deoxyribonucleic acid; TSDR, Treg-cell-specific demethylated region.